Figure 3.

Effect of DCA, LY294002 or combination of DCA and LY294002 on extracellular lactate concentration, key glycolysis-associated enzymes expression and HK-2 activation. The PASMCs were seeded into 25 cm² tissue culture flask at a density of 5×10⁵ cell/flask and cultured with RPMI-1640 complete culture medium for 16 h followed by serum starvation for 24 h. (A) The cells were then exposed to PDGF or 5 µM LY294002, DCA at 10 or 20 mM, and a combination of LY294002 and 10 mM DCA for 48 h prior to measurement of lactate concentration. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. control cells. (B) Cell lysates were extracted for PDK-1, PDH, HIF-1α and HK-2 expression analysis using western blot analysis. The representative change of one of the three experiments is presented, as all assays exhibited identical results. (C) HK-2 activation was analyzed with immune-fluorescent confocal microscopy. PDGF-stimulated PASMCs exhibited significant co-localization between HK-2 (green) and the mitochondria (red), giving a yellow pattern in the merged images. The cells treated with 20 mM DCA and the combination of LY294002 and 10 mM DCA demonstrated diffuse cytoplasmic staining of HK-2 (no colocalization of HK-2 to the mitochondria). C, control; DCA, dichloroacetate; PDGF, platelet-derived growth factor; L5, D10 and D20, cells treated with DCA at 5, 10 and 20 mM, respectively, following PDGF exposure; L5, cells treated with LY294002 at 5 µM following PDGF exposure; L5D10, cells treated in combination of 5 µM LY294002 and 10 mM dichloroacetate following PDGF exposure; PASMCs, pulmonary arterial smooth muscle cells; HK-2, hexokinase-2; PDK-1, pyruvate dehydrogenase kinase-1; PDH, pyruvate dehydrogenase; HIF-1α, hypoxia-inducible factor-1α; mito, mitochondria.